PCR based detection of tcdCΔ117 in Clostridium difficile infection identifies patients at risk for recurrence - A hospital-based prospective observational study.

OBJECTIVES: Increasing incidence and severity of Clostridium difficile infection (CDI) in the last decades has been attributed to the emergence of hypervirulent C. difficile strain PCR-ribotype 027 (RT027). Commercial multiplex real-time PCR tests allow the presumptive identification of RT027 by detecting a single-base deletion at nt117 in the tcdC gene (tcdCΔ117). The clinical usefulness of the detection of tcdCΔ117 is unclear. Therefore, we evaluated test performance and clinical association of the detection of tcdCΔ117 in patients with CDI in a prospective observational study conducted in a tertiary care hospital in Germany. METHODS: From June to October 2015, stool from all patients with suspected CDI was tested for C. difficile according to ESCMID guidelines. C. difficile was cultured from positive samples and ribotyping was performed. Clinical data were collected prospectively from all C. difficile positive patients. RESULTS: From 1121 tested stool samples 107 patients with CDI were included in the study. TcdCΔ117 was detected in 18 (16.8%) of these patients. Multivariable logistic regression analysis revealed an independent association of detection of tcdCΔ117 with a further episode of CDI (OR 14.6; 95% CI 3.6-58.3; p < 0.001) and death within 30 days of the positive test (OR 5.1; 95% CI 1.0-25.7; p = 0.046). As follow up data are limited, it remains unclear, whether the further episode of CDI was due to tcdCΔ117 (recurrence) or another type. CONCLUSION: In our setting, PCR-based detection of tcdCΔ117 identified patients at risk for recurrent CDI and increased mortality and thus may guide therapeutic choices in CDI patients at the time of diagnosis.

On chip quadruplex priming amplification for quantitative isothermal diagnostics.

Nucleic acid testing is a common technique for medical diagnostics. For example, it is used to detect HIV treatment failure by monitoring viral load levels. Quadruplex Priming Amplification (QPA) is an isothermal nucleic acid amplification technique that requires little power and few chemical reagents per assay, all features that make QPA well suited for point-of-care (POC) diagnostics. The QPA assay can be further optimized by integrating it with microfluidic devices that can automate and combine multiple reaction steps and reduce the quantity and cost of reagents per test. In this study, a real-time, exponential QPA reaction is demonstrated for the first time in a microfluidic chip, where the reaction was not inhibited and supported performance levels comparable to a commercially-available, non-microfluidics setup.

[Nontuberculous mycobacterial infections]. / Infektionen mit nichttuberkulösen Mykobakterien.

Nontuberculous mycobacterial (NTM) are found ubiquitously in the environment and are usually of low pathogenicity. Infection occurs via inhalation of aerosols, and some species may cause severe infections. The incidence of NTM infections is rising worldwide. The risk of developing NTM disease depends on the susceptibility of the host as well as the frequency and duration of exposure. In addition to congenital immune deficiencies and immunosuppressive therapy, structural lung and systemic diseases, including rheumatoid arthritis (RA), are associated with an increased risk for NTM infections. The immune response to NTM is complex and relies on the interplay between professional phagocytes and lymphoid cells. This interplay is concerted by three key cytokines: interleukin-12 (IL-12), tumor necrosis factor-α (TNF-α), and interferon-γ (IFN-γ). Targeted immunotherapies, e. g., treatment with TNF inhibitors, interfere with these essential pathways and increase the risk of NTM infection significantly. This review focuses on the relationship between the immune response to NTM and intrinsic and iatrogenic dispositions for NTM infection, with an emphasis on RA.

Comparative evaluation of two fully-automated real-time PCR methods for MRSA admission screening in a tertiary-care hospital.

We evaluated two fully-automated real-time PCR systems, the novel QIAGEN artus MRSA/SA QS-RGQ and the widely used BD MAX MRSA assay, for their diagnostic performance in MRSA admission screening in a tertiary-care university hospital. Two hundred sixteen clinical swabs were analyzed for MRSA DNA using the BD MAX MRSA assay. In parallel, the same specimens were tested with the QIAGEN artus MRSA/SA QS-RGQ. Automated steps included lysis of bacteria, DNA extraction, real-time PCR and interpretation of results. MRSA culture was additionally performed as a reference method for MRSA detection. Sensitivity values were similar for both assays (80 %), while the QIAGEN artus MRSA/SA QS-RGQ reached a slightly higher specificity (95.8 % versus 90.0 %). Positive (PPVs) and negative predictive values (NPVs) were 17.4 % and 99.4 % for the BD MAX MRSA assay and 33.3 % and 99.5 % for the QIAGEN artus MRSA/SA QS-RGQ, respectively. Total turn-around time (TAT) for 24 samples was 3.5 hours for both assays. In conclusion, both assays represent reliable diagnostic tools due to their high negative predictive values, especially for the rapid identification of MRSA negative patients in a low prevalence MRSA area.

Autoinfection as a cause of postpartum subdural empyema due to Mycoplasma hominis.

Mycoplasma hominis is a commensal of the genitourinary tract, which is infrequently associated with urogenital infections. Extra-urogenital infections due to M. hominis are rare. Here, we report an unusual case of M. hominis subdural empyema in a woman occurring shortly after delivery. The patient presented with symptoms suggestive of bacterial meningitis. Spinal imaging revealed a subdural empyema that required neurosurgical intervention. Cultures from intraoperatively obtained biopsies identified M. hominis as the causative pathogen. The patient was treated with oral moxifloxacin for 4 weeks resulting in the resolution of the spinal lesion. The subdural empyema was presumably caused by a contaminated epidural blood patch performed with the patient's own blood during an episode of transient M. hominis bacteremia after delivery. The blood patch was indicated for the treatment of cerebrospinal fluid leakage, which had occurred after epidural anesthesia. Our findings highlight the significance of transient M. hominis bacteremia after delivery and implicate that M. hominis should be considered as a causative agent of extra-genitourinary tract infections particularly during the postpartum period or after genitourinary manipulation.

Impact of bulge loop size on DNA triplet repeat domains: Implications for DNA repair and expansion.

Repetitive DNA sequences exhibit complex structural and energy landscapes, populated by metastable, noncanonical states, that favor expansion and deletion events correlated with disease phenotypes. To probe the origins of such genotype-phenotype linkages, we report the impact of sequence and repeat number on properties of (CNG) repeat bulge loops. We find the stability of duplexes with a repeat bulge loop is controlled by two opposing effects; a loop junction-dependent destabilization of the underlying double helix, and a self-structure dependent stabilization of the repeat bulge loop. For small bulge loops, destabilization of the underlying double helix overwhelms any favorable contribution from loop self-structure. As bulge loop size increases, the stabilizing loop structure contribution dominates. The role of sequence on repeat loop stability can be understood in terms of its impact on the opposing influences of junction formation and loop structure. The nature of the bulge loop affects the thermodynamics of these two contributions differently, resulting in unique differences in repeat size-dependent minima in the overall enthalpy, entropy, and free energy changes. Our results define factors that control repeat bulge loop formation; knowledge required to understand how this helix imperfection is linked to DNA expansion, deletion, and disease phenotypes.

DNA meter: Energy tunable, quantitative hybridization assay.

We describe a novel hybridization assay that employs a unique class of energy tunable, bulge loop-containing competitor strands (C*) that hybridize to a probe strand (P). Such initial "pre-binding" of a probe strand modulates its effective "availability" for hybridizing to a target site (T). More generally, the assay described here is based on competitive binding equilibria for a common probe strand (P) between such tunable competitor strands (C*) and a target strand (T). We demonstrate that loop variable, energy tunable families of C*P complexes exhibit enhanced discrimination between targets and mismatched targets, thereby reducing false positives/negatives. We refer to a C*P complex between a C* competitor single strand and the probe strand as a "tuning fork," since the C* strand exhibits branch points (forks) at the duplex-bulge interfaces within the complex. By varying the loop to create families of such "tuning forks," one can construct C*P "energy ladders" capable of resolving small differences within the target that may be of biological/functional consequence. The methodology further allows quantification of target strand concentrations, a determination heretofore not readily available by conventional hybridization assays. The dual ability of this tunable assay to discriminate and quantitate targets provides the basis for developing a technology we refer to as a "DNA Meter." Here we present data that establish proof-of-principle for an in solution version of such a DNA Meter. We envision future applications of this tunable assay that incorporate surface bound/spatially resolved DNA arrays to yield enhanced discrimination and sensitivity.

Energy landscapes of dynamic ensembles of rolling triplet repeat bulge loops: implications for DNA expansion associated with disease states.

DNA repeat domains can form ensembles of canonical and noncanonical states, including stable and metastable DNA secondary structures. Such sequence-induced structural diversity creates complex conformational landscapes for DNA processing pathways, including those triplet expansion events that accompany replication, recombination, and/or repair. Here we demonstrate further levels of conformational complexity within repeat domains. Specifically, we show that bulge loop structures within an extended repeat domain can form dynamic ensembles containing a distribution of loop positions, thereby yielding families of positional loop isomers, which we designate as "rollamers". Our fluorescence, absorbance, and calorimetric data are consistent with loop migration/translocation between sites within the repeat domain ("rollamerization"). We demonstrate that such "rollameric" migration of bulge loops within repeat sequences can invade and disrupt previously formed base-paired domains via an isoenthalpic, entropy-driven process. We further demonstrate that destabilizing abasic lesions alter the loop distributions so as to favor "rollamers" with the lesion positioned at the duplex/loop junction, sites where the flexibility of the abasic "universal hinge" relaxes unfavorable interactions and/or facilitates topological accommodation. Another strategic siting of an abasic site induces directed loop migration toward denaturing domains, a phenomenon that merges destabilizing domains. In the aggregate, our data reveal that dynamic ensembles within repeat domains profoundly impact the overall energetics of such DNA constructs as well as the distribution of states by which they denature/renature. These static and dynamic influences within triplet repeat domains expand the conformational space available for selection and targeting by the DNA processing machinery. We propose that such dynamic ensembles and their associated impact on DNA properties influence pathways that lead to DNA expansion.

Yersinia pseudotuberculosis bloodstream infection and septic arthritis: case report and review of the literature.

Yersinia pseudotuberculosis belongs to the family Enterobacteriaceae and is known to cause enterocolitis, terminal ileitis, pseudoappendicitis, erythema nodosum, reactive polyarthritis, and, occasionally, bloodstream infections. Here, we report the first case of bacteremia and septic arthritis in a patient without obvious risk factors and review all of the published cases of Y. pseudotuberculosis bloodstream infections.

Azole-resistant invasive aspergillosis in a patient with acute myeloid leukaemia in Germany.

We report the first culture-proven case of invasive aspergillosis (IA) caused by azole-resistant Aspergillus fumigatus in a patient with acute myeloid leukaemia in Germany. IA presented as breakthrough infection under posaconazole prophylaxis. Analysis of the resistance mechanism revealed the TR/L98H mutation in the cyp51A gene, which indicates an environmental origin of the strain. This case underscores the need for monitoring azole resistance in Aspergillus spp. and for routine susceptibility testing of moulds.

Energy crosstalk between DNA lesions: implications for allosteric coupling of DNA repair and triplet repeat expansion pathways.

Energy coupling between distal DNA domains may have profound regulatory consequences for biological processes, allowing for allosteric control of nucleic acid function. Repair of oxidative lesions at or near triplet repeat domains can enhance DNA expansion events that result in debilitating disease states. We report here position, distance, and lesion-dependent energy crosstalk between pairs of lesions in a triplet repeat bulge loop and an adjacent duplex domain. We discuss the implications of such coupled communication between lesions in distal loop and duplex domains for lesion repair and DNA expansion associated with diseases.

Heat Capacity Changes (ΔCp) for Interconversions between Differentially-Ordered DNA States within Physiological Temperature Domains: Implications for Biological Regulatory Switches.

Knowledge of differences in heat capacity changes (ΔCp) between biopolymer states provides essential information about the temperature dependence of the thermodynamic properties of these states, while also revealing insights into the nature of the forces that drive the formation of functional and dysfunctional biopolymer "order." In contrast to proteins, for nucleic acids there is a dearth of direct experimental determination of this information-rich parameter, a deficiency that compromises interpretations of the ever-increasing thermodynamic analyses of nucleic acid properties; particularly as they relate to differential nucleic acid (meta)stability states and their potential biological functions. Here we demonstrate that such heat capacity differences, in fact, exist not only between traditionally measured native to fully unfolded (assumed "random coil") DNA states, but also between competing order-to-order transformations. We illustrate the experimental approach by measuring the heat capacity change between "native"/ordered, sequence homologous, "isomeric" DNA states that differ in conformation but not sequence. Importantly, these heat capacity differences occur within biologically relevant temperature ranges. In short, we describe a new and general method to measure the value of such heat capacity differences anywhere in experimentally accessible conformational and temperature space; in this case, between two metastable bulge loop states, implicated in DNA expansion diseases, and their competing, fully paired, thermodynamically more stable duplex states. This measurement reveals a ΔCp of 61 ± 7 cal molbp -1 K -1. Such heat capacity differences between competing DNA "native" ensemble states must be considered when evaluating equilibria between different DNA "ordered" conformations, including the assessment of the differential stabilizing forces and potential biological functions of competing DNA "structured" motifs.

DNA repair and DNA triplet repeat expansion: the impact of abasic lesions on triplet repeat DNA energetics.

Enhanced levels of DNA triplet expansion are observed when base excision repair (BER) of oxidative DNA base damage (e.g., 8-oxo-dG) occurs at or near CAG repeat sequences. This observation suggests an interplay between processing mechanisms required for DNA repair and expansion pathways that yield genotypes associated with many neurological/developmental disorders. It has been proposed that DNA expansion involves the transient formation within the triplet repeat domains of non-native slipped DNA structures that are incorrectly processed by the BER machinery of repair during DNA synthesis. We show here that replacement within a triplet repeat bulge loop domain of a guanosine residue by an abasic site, the universal BER intermediate, increases the population of slipped/looped DNA structures relative to the corresponding lesion-free construct. Such abasic lesion-induced energetic enhancement of slipped/looped structures provides a linkage between BER and DNA expansion. We discuss how the BER machinery of repair may be influenced by abasic-induced energetic alterations in the properties of regions proximal to and/or within triplet repeat domains, thereby potentially modulating levels of DNA expansion.

Dynamic DNA Energy Landscapes and Substrate Complexity in Triplet Repeat Expansion and DNA Repair.

DNA repeat domains implicated in DNA expansion diseases exhibit complex conformational and energy landscapes that impact biological outcomes. These landscapes include ensembles of entropically driven positional interchanges between isoenergetic, isomeric looped states referred to as rollamers. Here, we present evidence for the position-dependent impact on repeat DNA energy landscapes of an oxidative lesion (8oxodG) and of an abasic site analogue (tetrahydrofuran, F), the universal intermediate in base excision repair (BER). We demonstrate that these lesions modulate repeat bulge loop distributions within the wider dynamic rollamer triplet repeat landscapes. We showed that the presence of a lesion disrupts the energy degeneracy of the rollameric positional isomers. This lesion-induced disruption leads to the redistribution of loop isomers within the repeat loop rollamer ensemble, favoring those rollameric isomers where the lesion is positioned to be energetically least disruptive. These dynamic ensembles create a highly complex energy/conformational landscape of potential BER enzyme substrates to select for processing or to inhibit processing. We discuss the implications of such lesion-induced alterations in repeat DNA energy landscapes in the context of potential BER repair outcomes, thereby providing a biophysical basis for the intriguing in vivo observation of a linkage between pathogenic triplet repeat expansion and DNA repair.

Calorimetry of proteins and nucleic acids.

The availability of sensitive calorimetric instrumentation has led to a considerable increase in thermodynamic studies of proteins, nucleic acids, and their interactions. This article reviews some of the recent contributions of calorimetry to characterizing the thermodynamic origins of protein and nucleic acid stability and conformational preferences, as well as the interactions of proteins with each other, with small molecules, and with nucleic acids.

The thermodynamics of DNA structures that contain lesions or guanine tetrads.

It is becoming increasingly apparent that energetic as well as structural information is required to develop a complete appreciation of the critical interrelationships between structure, energetics, and biological function. Motivated by this recognition, we have reviewed in this article the current state of the thermodynamic databases associated with lesion-containing DNA duplexes and DNA quadruplexes, while highlighting important considerations concerning the methods used to obtain the requisite data.

The macrophage-induced gene (mig) of Mycobacterium avium encodes a medium-chain acyl-coenzyme A synthetase.

The macrophage-induced gene (mig) of Mycobacterium avium has been associated with virulence, but the functions of the gene product were still unknown. Here we have characterized the Mig protein by biochemical methods. A plasmid with a histidine-tagged fusion protein was constructed for expression in Escherichia coli. Mig was detected as a 60 kDa protein after expression and purification of the recombinant gene product. The sequence of the fusion gene and of the parent gene in M. avium were reexamined. This confirmed that the mig gene encodes a 550 amino acid protein (58 kDa) instead of a 295 amino acid protein (30 kDa) as predicted before. The 550 amino acid Mig exhibits a high degree of homology to bacterial acyl-CoA synthetases. Two artificial 30 kDa derivatives of Mig were expressed and purified as histidine-tagged fusion proteins in E. coli. These proteins and the 58.6 kDa histidine-tagged Mig protein were analysed for activity with an acyl-CoA synthetase assay. Among the three investigated proteins, only the 58.6 kDa Mig exhibited detectable activity as an acyl-CoA synthetase (EC 6.2.1.3) with saturated medium-chain fatty acids, unsaturated long-chain fatty acid and some aromatic carbon acids as substrates. Enzymatic activity could be inhibited by 2-hydroxydodecanoic acid, a typical inhibitor of medium-chain acyl-CoA synthetases. We postulate a novel medium-chain acyl-CoA synthetase motif. We have investigated the biochemical properties of Mig and suggest that this enzyme is involved in the metabolism of fatty acid during mycobacterial survival in macrophages.

Thermodynamics of an intramolecular DNA triple helix: a calorimetric and spectroscopic study of the pH and salt dependence of thermally induced structural transitions.

We have characterized thermodynamically the melting transitions of a DNA 31-mer oligonucleotide (5'-GAAGAGGTTTTTCCTCTTCTTTTTCTTCTCC-3') which is designed to fold into an intramolecular triple helix. The first 19 residues fold back on themselves to form an antiparallel Watson-Crick hairpin duplex with a T5 loop. The 3'-terminal seven residues, which are connected to the Watson-Crick hairpin duplex by a second T5 loop, form Hoogsteen interactions in the major groove of the Watson-Crick hairpin. From ultraviolet (UV) melting studies we find that the 31-mer exhibits either one or two transitions, depending on solution conditions. We use pH- and temperature-dependent circular dichroism (CD) to assign the initial and final states associated with each transition. We find that the disruption of the Hoogsteen hairpin is accompanied by a release of protons and an uptake of sodium ions while the disruption of the Watson-Crick hairpin is accompanied by a release of sodium ions with no change in protonation state. From these data, we construct a phase diagram for this intramolecular DNA triple helix as a function of pH, sodium ion concentration, and temperature. We characterize the energetics of each transition using a van't Hoff analysis and differential scanning calorimetry (DSC). Significantly, the DSC data provide a model-independent thermodynamic characterization of the thermally induced transitions of this triplex. By combining the spectroscopic and calorimetric data, we develop a semi-empirical model which describes the state of the 31-mer as a function of pH, sodium ion concentration, and temperature. With this model we successfully predict characteristics of the 31-mer, which are beyond the data which are used in establishing the model (for example, the salt dependence of the apparent pKa of the Hoogsteen strand). This semi-empirical model may serve as a prototype for developing a method to predict the phase diagrams of intramolecular triple helix systems.

Energetics of lesion recognition by a DNA repair protein: thermodynamic characterization of formamidopyrimidine-glycosylase (Fpg) interactions with damaged DNA duplexes.

As part of an overall effort to map the energetic landscape of the base excision repair pathway, we report the first thermodynamic characterization of repair enzyme binding to lesion-containing duplexes. Isothermal titration calorimetry (ITC) in conjunction with spectroscopic measurements and protease protection assays have been employed to characterize the binding of Escherichia coli formamidopyrimidine-glycosylase (Fpg), a bifunctional repair enzyme, to a series of 13-mer DNA duplexes. To resolve energetically the binding and the catalytic events, several of these duplexes are constructed with non-hydrolyzable lesion analogs that mimic the natural 8-oxo-dG substrate and the abasic-like intermediates. Specifically, one of the duplexes contains a central, non-hydrolyzable, tetrahydrofuran (THF) abasic site analog, while another duplex contains a central, carbocyclic substrate analog (carba-8-oxo-dG). ITC-binding studies conducted between 5.0 degrees C and 15.0 degrees C reveal that Fpg association with the THF-containing duplex is characterized by binding free energies that are relatively invariant to temperature (deltaG approximately -9.5 kcalmol(-1)), in contrast to both the reaction enthalpy and entropy that are strongly temperature-dependent. Complex formation between Fpg and the THF-containing duplex at 15 degrees C exhibits an unfavorable association enthalpy (deltaH=+7.5 kcalmol(-1)) that is compensated by a favorable association entropy (TdeltaS=+17.0 kcalmol(-1)). The entropic nature of the binding interaction, coupled with the large negative heat capacity (deltaC(p)=-0.67 kcaldeg(-1)mol(-1)), is consistent with Fpg complexation to the THF-containing duplex involving significant burial of non-polar surface areas. By contrast, under the high ionic strength buffer conditions employed herein (200 mM NaCl), no appreciable Fpg affinity for the carba-8-oxo-dG substrate analog is detected. Our results suggest that initial Fpg recognition of a damaged DNA site is predominantly electrostatic in nature, and does not involve large contact interfaces. Subsequent base excision presumably facilitates accommodation of the resulting lesion site into the binding pocket, as the enzyme interaction with the THF-containing duplex is characterized by high affinity and a large negative heat capacity change. Our data are consistent with a pathway in which Fpg glycosylase activity renders the base excision product a preferred ligand relative to the natural substrate, thereby ensuring the fidelity of removing highly reactive and potentially mutagenic abasic-like intermediates through catalytic elimination reactions.

Phagocytosis and killing of Mycobacterium avium complex by human neutrophils.

Organisms belonging to the Mycobacterium avium complex (MAC) cause life-threatening bacteremia in immunocompromised patients. Monocytes and macrophages are thought to be responsible for ingestion and killing of MAC. However, it has been suggested that neutrophils may play a role in the early immune response to MAC infection. Here, neutrophils in autologous plasma were incubated (at 0 and 37 degrees C) with M. avium labeled with Auramine O, a potent fluorochrome. Neutrophil phagocytosis was measured by flow cytometry. Neutrophils incubated at 37 degrees C showed an increase in fluorescence over time with a maximum at 15 min, whereas neutrophils on ice showed no time-dependent increase in FL1. At 15 min Fl 1 at 37 degrees C was twice as high as FL1 at 0 degrees C. Examination under the fluorescent microscope showed multiple intracellular fluorescent mycobacteria. Results in nine independent experiments showed time-dependent decrease of colony-forming units in neutrophil-associated live M. avium. Significant killing was observed within 30 min and was complete by 120 min. Observation by electron microscopy clearly confirmed the presence of intraphagosomal MAC, both intact and with evidence of degradation. These data demonstrate that MAC is rapidly phagocytized and killed by human neutrophils. The newly established flow cytometry method should be useful in further studies of neutrophil function and of the role of G-CSF and other cytokines in MAC disease.